Angiotensin II is a vasoconstrictor and a hypertensive peptidethat binds to the AT1 receptor and, through both direct andindirect mechanisms, induces salt reabsorption. Also, angiotensinII is thought to be a profibrotic and proproliferative peptide;abundant evidence now suggests that angiotensin II can stimulatecytokines and oxygen radicals, inducing an inflammatory response(1,2). Indeed, so much has been written about angiotensin-convertingenzyme (ACE) and angiotensin II that it is legitimate to askwhat new can be found concerning actions of the renin-angiotensinsystem (RAS). This review shows that, rather than being a subjectwithout novelty, the ability to genetically manipulate micegives scientists the power to investigate the many physiologicroles of angiotensin II with a level of understanding not previouslyavailable. At last, scientific tools are available to test someof the hypotheses and dogmas concerning RAS action. Perhapsyou are not convinced and think that, in a system that has seenthe publication of angiotensinogen, renin, and ACE knockoutmice, how could even the powerful technology of mouse genetictargeting provide novel insight? Answering precisely this questionis the subject of the review.
The technique of creating a knockout mouse is now widely appreciated(3). Cultured embryonic stem cells are manipulated by targetedhomologous recombination to induce virtually any genetic changethat can be imagined. The most common use of this method hasbeen to eliminate a gene that, in a mice that are homozygousfor the mutation, results in a knockout animal. In such models,no protein is made during the entire embryonic and extraembryoniclife of the animal. The resulting phenotype is often dramaticand at any rate gives great insight into the functional roleof a protein as assessed in a mammal that is not fundamentallydifferent from humans. For example, mice that lack angiotensinogen,ACE, or all AT1 receptors have a similar phenotype (4–9).These animals show a reduction of systolic BP in excess of 35mmHg. They are unable to produce a concentrated urine much above1000 mOsm/L. They have elevated serum and urinary potassiumconcentrations. They are anemic, and they present with a renallesion characterized by renal medullary thinning associatedwith expansion of the renal pelvis. ACE knockout mice also havedefective male fertility as a result of the absence of the testisisozyme of ACE. Published work suggests that this reproductivedefect is not found in angiotensinogen knockout mice, suggestingan important substrate for ACE other than angiotensin I (10).
Although knockout mice are a wonderful methodologic advance,this technique has some disadvantages. For instance, the completelack of protein production is different from a human who takesa pharmaceutical to inhibit that protein. Indeed, the more oneworks with knockout mice, the more one is aware that the totalgenetic abrogation of a protein represents a truly extreme phenotype.The most common human diseases typically do not result fromthe complete absence of a particular protein.
The power of targeted recombination in embryonic stem cellsextends beyond the ability to create a simple knockout model.For example, the research group of Smithies and colleagues (5)used gene targeting to create a series of genetically alteredmice that bear zero to four copies of the angiotensinogen gene.This celebrated study used gene targeting to either eliminateor duplicate the angiotensinogen gene and showed that therewas a gene dosage effect in which every additional copy of theangiotensinogen gene beyond one was responsible for an 8-mmHgrise in BP. These data, combined with human studies, suggestthat genetic variability in angiotensinogen can account forsome genetic differences in human BP (11,12).
In addition to gene elimination or duplication, targeted homologousrecombination can create precise mutations within a protein.For example, our group used such an approach to study ACE. Thisenzyme is a single polypeptide chain but contains two zinc bindingsites and two separate and independent catalytic domains (13).Although angiotensin I is a good substrate for both of the twocatalytic sites of ACE, other peptide substrates show distinctpreferences for only one of the ACE catalytic sites. An exampleis the ACE substrate acetyl-SDKP that has been implicated asan antifibrotic and a bone marrow–suppressive peptide(14,15). Acetyl-SDKP is effectively hydrolyzed only by the N-terminalcatalytic domain of ACE; ACE inhibition is associated with anelevation in blood levels of this peptide (16). As discussed,ACE null (knockout) animals have anemia characterized by hematocritsapproximately 20% lower than that in wild-type mice. One hypothesissuggested that the anemia was a consequence of acetyl-SDKP build-upin the absence of ACE activity. To test this, our group introducedpoint mutations into the ACE gene that eliminated the abilityof the N-terminal ACE catalytic domain to bind zinc and thusrendered this domain catalytically inactive (Figure 1) (17).The result was a mouse model called ACE 7/7, which makes normalamounts of an altered ACE protein that has only one catalyticdomain. In the absence of the N-terminal domain, these animalshave elevated serum levels of acetyl-SDKP but a normal BP asa result of the ability of the unaltered C-terminal ACE catalyticdomain to effectively hydrolyze angiotensin I. Study of thesemice found that they had no evidence of anemia. Thus, this isan example in which genetic targeting in mice produced an alteredprotein, as opposed to the complete elimination of that protein.This particular model allowed study of the physiologic effectsof acetyl-SDKP accumulation in a setting free of the secondaryeffects of low BP, and ultimately it disproved the suggestedhypothesis for the actions of acetyl-SDKP. Even with this study,it remains unclear why animals that lack all ACE (ACE knockoutmice) are anemic. Most likely, the anemia reflects the inabilityto generate angiotensin II (as opposed to another peptide) becausethe infusion of angiotensin II into ACE null mice is associatedwith correction of the anemia (18).
Figure 1. (A) In a wild-type animal, angiotensin-converting enzyme (ACE) is a single polypeptide chain with two zinc (Zn) binding sites and two catalytic domains. The ACE.7 mutation introduced point mutations into the ACE gene that changed the amino acid sequence of the N-terminal Zn binding site from HEMGH to KEMGK. This new sequence is unable to bind zinc, eliminating catalytic activity in the N-terminal domain. ACE 7/7 mice are homozygous for the ACE.7 mutation. The ACE made by these mice has catalytic activity only in the C-terminal domain. (B) A rabbit anti-ACE antibody was used in Western blot analysis to evaluate ACE protein expression in wild-type (wt), ACE 7/7, and ACE 1/7 mice. ACE 1/7 animals are compound heterozygotes in which the 1 allele is null for ACE expression. ACE 7/7 mice express ACE levels equivalent to wt animals. In contrast, ACE 1/7 mice have reduced ACE protein expression.
If one views ACE null mice as expressing an extreme phenotype,then it would be useful to have mouse models with a less severegenetic change. One approach might be to selectively eliminateACE expression in a particular organ such as the kidney (Figure 2).This is technologically feasible using gene targeting butprobably would not yield a phenotype in the resulting mice.ACE is widely distributed in animal tissues; large amounts ofACE are made by vascular endothelium, the kidney, areas of thegut, and activated macrophages and in parts of the brain. Giventhe highly regulated expression of renin, it seemed unlikelythat the elimination of ACE expression in one target organ wouldhave much effect. Our group has selected a different approach:To create a mouse model devoid of ACE activity, except for selectedorgans engineered to express this protein (Figure 2). Modelsof this type can be created by genetic manipulation of the promoterregion of the ACE gene (the portion of the gene that regulatestissue and temporal expression). For example, consider the mousemodel that is termed ACE.3, in which genetic engineering wasused to position the albumin promoter in place of the naturalACE promoter (Figure 2B) (19). In these mice, ACE expressionis controlled not by the ACE promoter but by the engineeredalbumin promoter, leading to ACE protein expression by hepatocyteswithin the liver. Because the coding portions of the ACE genewere not modified, the protein was transported to the hepatocytecell surface, similar to its typical localization in endothelium.However, as the albumin promoter is active only in hepatocytes(and not in endothelium), this model resulted in mice with veryrestricted ACE expression. For example, wild-type mice normallyproduce large amounts of ACE in the lung as a result of thehigh content of endothelium within this tissue (Figure 3). Incontrast, ACE.3 mice produced no ACE in the lung or by any endotheliumthroughout the mouse. Only in the kidney was there aberrantlow-level recognition of the modified ACE promoter, leadingto renal ACE levels approximately 15% those of wild-type mice.The low-level ACE found in kidney was present in the proximaltubule and probably resulted from the use of a man-made albuminpromoter to control ACE expression. However, even in the kidney,vascular expression of ACE was not present. Thus, a mouse modelin which the widespread distribution of ACE in endothelium andepithelium was replaced by localized ACE expression on the surfaceof hepatocytes was created.
Figure 2. (A) In a wt mouse, ACE is widely distributed. In contrast, a knockout mouse that is null for all ACE expression represents an extreme phenotype in that this animal makes no ACE during its entire lifetime. One way to approach a mouse model with selective expression of ACE is to eliminate genetically the protein from a particular organ such as the kidney. However, given the wide distribution of ACE, such a model seemed unlikely to give a phenotype. The approach that our group has adopted is to functionally replace the endogenous ACE promoter with another, tissue-specific promoter. In this way, expression of ACE is directed to the small subset of tissues that recognize the new promoter. (B) The genetic construct used to create the ACE.3 mutation. Arrows indicate the endogenous somatic and testis promoters. The wt ACE allele was modified by the insertion of a neomycin cassette followed by the albumin promoter between the endogenous somatic ACE promoter and the transcribed portions of the gene. In this way, the effects of the endogenous ACE promoter are blocked and control of the gene is directed by the albumin promoter.
Figure 3. Tissue distribution of ACE in ACE 3/3 mice. Tissues from a wt mouse (+/+) and an ACE 3/3 mouse (–/–) were fixed in formalin and prepared by immunostaining with a rabbit polyclonal anti-ACE antibody. ACE is indicated as a brown pigment. (A and B) Sections of liver and show that, whereas a wt mouse makes very little ACE in this tissue (A), the ACE 3/3 mouse expresses abundant hepatic ACE (B) and localizes the protein on the hepatocyte cell surface (B, insert). In contrast, wt mice have abundant ACE in the lung (C), but ACE 8/8 mice express no ACE in lung (D). In the kidney, wt mice make abundant ACE in the straight portion of the proximal tubule (E). In ACE 3/3 mice, renal ACE is reduced to approximately 14% that of wt levels (F). In a wt mouse, an abundant source of ACE is vascular endothelium (G, arrow) and the vascular adventitia. As seen in H, ACE 3/3 mice have no vascular ACE expression by either endothelium or the adventitia. These data show that ACE expression in ACE 3/3 mice was genetically transferred from the endothelium and the kidney to the liver.
One advantage of this approach is that compound heterozygousmice can easily be created through simple breeding. For instance,our group refers to ACE knockout mice as ACE.1; a homozygousknockout mouse has an ACE genotype called ACE 1/1 to indicatethat both ACE alleles contain the ACE.1 mutation (Table 1).These animals are null for all ACE expression. Likewise, a mousehomozygous for the ACE mutation that targets expression to theliver has a genotype termed ACE 3/3. A mouse with point mutationsthat inactivate the ACE N-terminal catalytic domain is termedACE 7/7. These numbers merely refer to the order in which themouse models were created, but they are useful in signifyingthe ACE genotype. For instance, an ACE heterozygous mouse withone ACE.1 allele and one wild-type allele is termed wt/1. Ifthis animal is mated with an ACE 3/3 mouse, then half of theoffspring will have the genotype ACE 1/3. In these mice, the"1" ACE allele is null, whereas the "3" ACE allele directs ACEexpression to the liver. Because of the null allele, ACE 1/3mice have half of the hepatic, renal, and plasma levels of ACEfound in ACE 3/3 mice (20). As compared with wild-type mice,ACE 1/3 mice have only 7% normal renal ACE activity.
Table 1. Important characteristics of mouse lines with genetic changes to the ACE genea
This promoter-swapping technique was used to create a seriesof mice with restricted patterns of ACE expression (Table 1).Rather than discuss each model individually, the major conclusionsobtained from the combined data are presented below.
Mice that lack all ACE have a systolic BP of approximately 73mmHg as opposed to a wild-type mouse with a systolic BP of approximately110 mmHg (Figure 4). This marked reduction in the absence ofall ACE belies that mice (and probably humans) are very tolerantof marked changes in tissue patterns and overall levels of ACEexpression. For example, ACE 1/7 mice, which express only halfof normal levels of an ACE protein that lacks enzymatic activityin one of the two ACE catalytic domains (the N-terminal domain),have a normal BP (17). ACE 1/3 mice, animals with no endothelialexpression of ACE and renal levels approximately 7% those ofwild-type mice, also have a normal BP (20). Even an animalsthat are engineered to express ACE only in myocardium and lungsmooth muscle (ACE 8/8) present with a BP not much differentfrom that of wild-type mice (21). The ability to tolerate widechanges in ACE activity was postulated through a computer-basedanalysis of BP control conducted by Smithies et al. (22). Thisgroup predicted that as ACE levels diminished, compensatoryupregulation of renin and angiotensin I levels would lead tohomeostatic maintenance of angiotensin II (and BP) until sucha point as ACE activity fell to such low levels as to overwhelmthe kidney’s ability to compensate. This conclusion ishighly consistent with what our group observed experimentally.In the ACE 1/3 mouse, normal BP is maintained by an elevationof plasma renin, plasma angiotensin I, and even plasma angiotensinII concentrations. ACE 1/3 mice have normal concentrations ofserum and urinary aldosterone. These data suggest that althoughthe plasma levels of angiotensin II are elevated, the end-organlevel of angiotensin II is precisely regulated to maintain anormal BP. More than just changes in ACE activity, studies ofgenetically manipulated mice show that animals are remarkablytolerant of different tissue patterns of ACE expression. Intotal, this work suggests that the proper way to view ACE isto apply the concept of "total body load" of ACE. As total bodyload diminishes, the kidney compensates through increased productionof renin. In a mouse, even modest total body loads of ACE positionedin tissues as diverse as either the liver or the heart are sufficientfor renal compensation and maintenance of normal BP. Only whenACE activity falls to very low levels can the RAS not achievecompensation. These conclusions apply to otherwise healthy animalsthat are maintained in a relatively disease- and stress-freelaboratory environment.
Figure 4. (A) BP was measured in conscious mice using a computer-controlled tail-cuff system that determines systolic BP (40). Mice were trained in the machine for several days before data were collected. The final data point for each mouse is the average of 80 measurements taken over 4 d. +, wt ACE allele; other strain designations are explained in Table 1. (B) Plasma renin activity was measured by assessing the conversion of rat angiotensinogen to angiotensin I during a normal salt diet and during salt deprivation. The designation 3/wt refers to a compound heterozygous mouse with one ACE allele targeting expression to the liver (the "3" allele) and one wt ACE allele. These mice are phenotypically similar to wt mice. ACE 1/3 are compound heterozygotes with one ACE null allele (the "1" allele) and one ACE 3 allele. Even with a normal salt diet, these mice upregulate renin activity to maintain a normal BP. In the absence of dietary salt, all animals increase renin expression. ACE 1/3 mice continue to maintain BP by a marked increase of plasma renin activity.
In addition to producing angiotensin II, ACE is thought to playa major role in the degradation of the vasodilator bradykinin.To study this, we bred ACE knockout mice with a different lineof mice that lack the bradykinin B2 receptor to generate double-knockoutmice (23). It is the B2 receptor that is thought to mediatethe cardiovascular effects of bradykinin (24). The experimentaldesign was to compare mice that lack only ACE with the double-knockoutmice that lack both ACE and the bradykinin B2 receptor. Thedouble-knockout mice had a phenotype identical to single ACEknockout mice (very low BP, underdevelopment of renal medulla,and inability to concentrate urine). Although this study wassomewhat incomplete in that it did not account for bradykininB1 receptors, it suggests that the main pathologic defect inACE null mice is a lack of angiotensin II and not a surplusof bradykinin.
What about the question of local versus systemic productionof angiotensin II? This discussion reflects the presence ofvarious components of the RAS throughout the body. In turn,this has engendered a lively discussion as to the importanceof local production of angiotensin II (tissue based) versusthe systemic generation of the peptide (25,26). Examining datafrom all of our models gives insight into this controversy.First, in both humans and mice, the majority of ACE activityis physically associated with endothelium and other tissuessuch as tubular epithelium in the kidney. Tissue ACE is sucha large percentage of the total body load that it is responsiblefor the majority of the conversion of angiotensin I to angiotensinII. However, total ACE levels are not in great excess, as indicatedby ACE wt/1 mice with roughly 60% wild-type ACE levels. Eventhis relatively modest reduction of ACE necessitates increasedangiotensin I production to maintain normal plasma levels ofangiotensin II and normal BP (18). In fact, animals can easilymaintain normal BP in the complete absence of all endothelialor renal expression of ACE. This is because, under basal conditions,there is both a tissue-fixed and a circulating pool of ACE.As the endothelial generation of angiotensin II diminishes,the kidney is able to compensate through upregulation of circulatingrenin, angiotensin I, and thus angiotensin II generation. Thus,our view of the controversy of local versus systemic generationof angiotensin II is that, at least for BP control, both ofthese systems contribute to normal regulation. In a normal animal,it is the final concentration of angiotensin II that is regulated;this peptide originates from both systemic and local production.If local production is genetically diminished, then systemicproduction is upregulated.
In the kidney, ACE is associated with vascular endothelium andproximal tubular epithelium, particularly the distal (S3) portionof the proximal tubule. We now have examined two strains ofmice with very low or no renal ACE. This is the ACE 1/3 mouse(7% normal renal ACE) and the ACE 8/8 mouse (no renal ACE) (20,21).In both strains, deprivation of water for 24 h resulted in urinaryconcentrations in excess of 3000 mOsm/L, which was indistinguishablefrom wild-type mice. Perhaps this is not surprising given thatthese animals retain a functional RAS with the ability to stimulatealdosterone production. However, it does indicate that the ACEwithin the kidney plays no irreplaceable physiologic role, atleast as measured by renal concentrating ability.
We studied ACE 1/3 mice after a 2-wk diet that totally lackedNaCl (21). In the absence of all exogenous salt, mice maximallyretained salt. Nonetheless, there is a small loss of urinarysalt and a consequent small reduction of BP. A typical wild-typemouse will reduce systolic BP by 5 mmHg or less. Under the extremestress of 2 wk without salt, we did observe a difference betweenthe ACE 1/3 mice and wild-type animals: There was a small butsignificant additional loss of urinary sodium by the ACE 1/3mice. This resulted in a greater reduction of systolic BP (approximately10 mmHg). Nonetheless, it was remarkable that ACE 1/3 mice wereable to tolerate easily this regimen and to maintain systolicBP in excess of 90 mmHg. An explanation for the remarkable behaviorof the ACE 1/3 mice is the elevation of plasma renin levels(Figure 4B). These data show that, under basal conditions, theACE 1/3 mice have an elevated renin level as compared with wild-typemice (it is this that maintains normal BP). In response to totalsalt deprivation, the RAS is activated and these mice respondwith much higher plasma renin levels than control animals. Thus,the ACE 1/3 mice dramatically demonstrate that, given a normalkidney and its compensatory ability, animals can tolerate amarked change in both the quantity and the tissue expressionpatterns of ACE. Under conditions of salt deprivation, the ACE1/3 mice maintain substantial homeostasis; it is only underextreme conditions that the selective lack of ACE expressionby endothelium and within the kidney seems at all deleterious.We end this portion of the discussion with a caveat that ourexperiments were performed with young mice that were held underlaboratory conditions. Clearly, the human response to the chronicinjuries that are typical of old age are different paradigms.Although the ACE 1/3 mice have not yet been tested in theseparadigms, they do underline the amazing ability of the RASto compensate given normal renal function. In a sense, thesedata support the idea that abnormalities in human BP must beassociated with some type of renal dysfunction.
Mice with altered tissue patterns of ACE expression provideinsight into the organ-specific generation and function of angiotensinpeptides. In fact, an evaluation of angiotensin peptide levelswas recently performed in mice that genetically lack ACE andin wild-type mice that were exposed to the ACE inhibitor lisinopril(27). These data proved that, in the mouse, ACE is the predominantpathway leading to angiotensin II formation. ACE is responsiblefor at least 90% of the conversion of angiotensin I to angiotensinII in the blood, kidney, heart, lung, and brain and at least77% in the adrenal. In fact, evaluation of angiotensin II peptidelevels in the kidneys of ACE null mice showed that this peptidewas reduced by >97% as compared with wild-type mice. Thesedata suggest that, at least in the mouse, chymase-like enzymesdo not play an important role in angiotensin II formation. Thesedata also completely refute the study by Wei et al. (28) reportingsimilar angiotensin I and angiotensin II levels in the organsof ACE knockout mice as compared with control animals. AngiotensinII is difficult to isolate under conditions that prevent theartificial conversion of angiotensin I (present in large amountsin ACE null mice) into angiotensin II. Our group took particularcare to homogenize freshly isolated organs in 4 mol/L guanidinethiocyanate to prevent this artifact. Data from our group documentingmarkedly reduced tissue angiotensin II levels in ACE knockoutmice seems very consistent with the striking reduction of BPmeasured by all groups who have studied ACE null mice (6,7).
An interesting aspect of the kidney is that wild-type mice haverenal levels of angiotensin II that are significantly higherthan those found in blood. This has engendered the idea thatrenal angiotensin II levels are the result of de novo formationin the kidney (29). Our data argue against de novo formationas being the major source of renal angiotensin II. ACE 8/8 micehave no ACE in renal tissue, instead having ACE within the heartand the blood. Despite no renal ACE, the kidney concentrationof angiotensin II averaged 130 fmol/g. This was >16 timeshigher than the concentration observed in the plasma and evenexceeded the level found in the lung of a wild-type mouse. Thehigh residual angiotensin II peptide concentrations presentin renal tissue that totally lacks tissue-bound ACE suggeststhat a significant percentage of total renal angiotensin IIpeptide levels must be due to absorption of the peptide fromthe blood.
Our group has worked hard to create a mouse with ACE expressionrestricted to the kidney. It is our belief that the evaluationof this model or even mouse lines with ACE expression limitedto different portions of the nephron will be informative asto the special role of the kidney in the maintenance of BP andelectrolyte balance. Although animal models previously discussedindicate that renal concentrating ability is retained in theabsence of renal ACE, it is important to remember that thesemodels have other compensating sources of ACE. Wild-type micedo contain considerable ACE in the brush border of the proximaltubule and in renal vascular endothelium. Our hope is that animalswith ACE expression restricted to the kidney will provide insightinto the local, renal generation of angiotensin II and the renaland systemic effects of this peptide.
My group prepared three mouse lines that were engineered suchthat control of the ACE gene was under promoters that were previouslydescribed as specifically active in the kidney. These are the glutamyl transpeptidase (-GT), the kidney androgen regulatedprotein (KAP), and the Tamm-Horsfall promoters. Although publishedliterature supported the use of these promoters in targetinggene expression to the kidney, our experience was very different(30–35). The KAP and Tamm-Horsfall promoters were inactiveand gave rise to mice that were similar in BP and renal structureto the ACE null mice previously discussed in this article. Evenan ACE 8/Tamm-Horsfall heterozygous animal, in which the "8"allele targets ACE to the heart and ensures normal renal tubulardevelopment, lacked renal expression of ACE (data not published).The -GT promoter did lead to small levels of ACE expressionwithin the kidney, but these levels were only approximately6% the normal ACE expression present in wild-type kidneys. Thecombination of low renal expression and the lack of ACE expressionin other organs of the animal resulted in the -GT mice havinga low BP equivalent to that of ACE null animals (data not published).
In one manner, these mice were different from an animal absolutelynull for all ACE expression. These new strains of mice continuedto express the testis isoform of ACE. Because of this, the micewere fully fertile, despite low BP. This compares to ACE nullmice, lacking testis ACE, in which fertility is markedly reducedin male mice. Our group now routinely uses mice that we termACE 4/4 (homozygous for the mutation in which the KAP promotercontrols ACE expression) as a substitute for ACE 1/1 null mice(36). Nonetheless, the lack of reliable renal-specific promoterswas a disappointment and a source of frustration for our group.The development of tools for reliably expressing proteins selectivelywithin the kidney is important. The lack of reliable renal-specificpromoters will hinder the genetic manipulation of mice and thephysiologic evaluation of protein expression in the kidney.
The role of ACE and angiotensin II within the heart has beenthe subject of much discussion. Some authors have suggestedthat the local generation of angiotensin II within cardiac tissuemay be deleterious, promoting pathologic development of cardiacfibrosis (37,38). To investigate this question, we made a mousein which the cardiac-specific -myosin heavy chain promoter waspositioned to control the ACE gene (21). Our hope was that thisanimal would make ACE selectively in cardiac tissue, thus focusingproduction of angiotensin II specifically within the heart.In fact, precisely this occurred in these mice called ACE 8/8.Whereas wild-type mice have virtually no ACE expression by themyocardium, the ACE 8/8 animals have extensive ACE enzyme locatedon the surface of cardiac myocytes in both the atria and theventricles. Ironically, vascular endothelium within the heartis completely absent of ACE expression. Although the -myosinheavy chain promoter is often thought to be specific for theheart, we found that the promoter also induced an unusual expressionpattern in the lung, where some vascular smooth muscle expressedACE. In addition, there was a patchy distribution of ACE withinthe lung parenchyma with the result that the lungs of ACE 8/8mice have approximately 40% normal ACE activity. In contrastto the lung, ACE 8/8 mice totally lack renal expression of ACE;these animals are null for all renal epithelial and vascularexpression. Indeed, endothelium throughout the animal produceno ACE.
We carefully evaluated cardiac levels of angiotensin II. Ourhope was that these animals would increase the cardiac concentrationof angiotensin II, and, in fact, we found levels of the peptidethat were 4.3-fold greater than those of wild-type mice. Althoughthe BP of the mice was near normal and renal concentrating abilitywas indistinguishable from wild-type mice, the ACE 8/8 animalsdefinitely were not normal; the death rate of the mice was markedlyincreased such that only 64% of the animals were alive 66 dafter birth (as opposed to 100% survival for wild-type mice).The increased mortality in this model was the result of twounusual findings: A marked increase in size of the atria andmarked cardiac electrical abnormalities characterized by lowvoltage and atrial fibrillation (Figure 5). However, to oursurprise, the ventricles of ACE 8/8 mice were in many ways normal.For example, histologic staining to identify collagen detectedno increase of ventricular fibrosis. Both echocardiography andintraventricular catheterization (with a Millar catheter) failedto show any marked abnormalities of ventricular function despitegreater than a fourfold increase of cardiac angiotensin II.Thus, this model questions the concept of an intrinsically deleteriouseffect of angiotensin II for myocardial function.
Figure 5. (A) Hearts of wt, ACE wt/8 heterozygous, and ACE 8/8 homozygous mice. The arrow indicates the markedly enlarged atria found in the ACE 8/8 mice. However, ventricular size is not different. (B) A typical electrocardiogram of a wt mouse. (C) An electrocardiogram from an ACE 8/8 mouse. ACE 8/8 mice consistently demonstrated reduced electrical amplitude of the QRS complex. Also, these mice lack P waves and have an abnormal rhythm, typical features of atrial fibrillation.
In contrast to the myocardial findings, the ACE 8/8 mice developedatrial enlargement between 2 and 3 wk of age through mechanismsthat are under intense study. Although work continues to helpus understand the phenotype of these mice, the findings in theACE.8 model underscore that genetic manipulation of mice isa powerful method to test accepted concepts of pathology anddisease etiology. For example, there is an extensive literatureimplicating angiotensin II as proinflammatory for disease processessuch as vascular injury and even atherosclerosis (2,39). Ourgroup is preparing mice that overexpress ACE by macrophages.Our hope is to use this and other mouse models to quantify preciselythe role of angiotensin II in vascular injury.
Our group has created mouse models in which ACE expression islimited to certain small subsets of tissue. This was in responseto the realization that a total ACE null mouse represents anextreme phenotype that, although very interesting, is intrinsicallylimited. Our new mouse models are examples of an approach tomanipulate gene promoters and restrict/rearrange protein expression.Some of what we found was contrary to previously held conceptsof tissue-specific RAS expression. This emphasizes the powerof our approach and of genetic manipulation of the mouse asa tool to dissect the complex role of the RAS. We hope thatthese mouse models will be a resource for many other laboratoriesinterested in the physiology and pathology of renal and cardiacdisease.
Acknowledgments
This work was supported by grants from the National Institutesof Health (DK39777, DK51445, and DK55503). H.D.X. is supportedby a fellowship from the Georgia affiliate of the National KidneyFoundation. K.F. is supported by a postdoctoral fellowship fromthe National Institutes of Health (F32 DK65410). S.F. is supportedby a postdoctoral fellowship from the National Kidney Foundation.
We acknowledge the help of Dr. Mario Capecchi, Department ofGenetics, University of Utah, and Dr. Pierre Corvol, Collegede France, Paris, France.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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glutamyl transpeptidase (
-myosin heavy chain promoter was positioned to control the ACE gene (21). Our hope was that this animal would make ACE selectively in cardiac tissue, thus focusing production of angiotensin II specifically within the heart. In fact, precisely this occurred in these mice called ACE 8/8. Whereas wild-type mice have virtually no ACE expression by the myocardium, the ACE 8/8 animals have extensive ACE enzyme located on the surface of cardiac myocytes in both the atria and the ventricles. Ironically, vascular endothelium within the heart is completely absent of ACE expression. Although the 
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Introduction
Knockout Mice: Advantages and...
